RT-PCR - basics (Jan/04/2005 )

Hello,
Hope everyone’s had a lovely festive season.
I’m meant to be starting some RT PCR. Never done it before.
Now, I have to check that the primers I’ve designed work. How is this done? How much RNA? (is 5 or 10 ug enough?)
Any tips of how to avoid major stuff ups?
General advice will be so appreciated.

Cheers
Vetticus.

Hi Vetticus,

For RT-PCR primer design, because RNA may be contaminated by DNA which can be coamplified with RNA, RT-PCR primers should span introns so that you can tell which PCR product is from DNA if there is contamination. To check if primers work, you have to first reverse transcribe your RNA (from a source that expresses your gene) into cDNA, and then do a PCR.

You can use 1-2 ug total cellular RNA for your RT reaction. If you're going to do one-step RT-PCR, use about 50 ng RNA as template.

Always include controls for RT and PCR reaction.

Work in a RNase free environment.

You have to also have primers for some housekeeping genes such as beta-actin, G3PDH and amplify them for RNA loading control.

Run your PCR for the cycles where the amplification is still in linear range.

These are what I can think of.

Good luck!

Hi,vetticus3. I think psulina has say most of the important things. I just
mention that you should remember to see the GC content of your amplification fragment. I spent more than one month to do RT-PCR of one gene. I can't get the reults just because the GC content of that gene is very high(71%).

Good luck.

Hi everyone
I'm quite in the same situation as vetticus3 and have a related question.
Primer design tips for real time suggest Tm to be 58-60 or at least over 50.
Why is that? How important is that? My Tm's are all around 55 and I guess that in any case I can set the temperature in the annealing step of the PCR to fit my primers.
Any advise will be more than welcomed.
thanks!